Tagged epitope protein transposable element

ABSTRACT

A transposable element is provided that as a 3′ and a 5′ end. The transposable element includes a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding and MHC epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element. In one embodiment, a transposable element is provided that has a 5′ and a 3′ end. The transposable element includes a 5′ loxP sequence 5′ of a nucleic acid encoding a selectable marker, a 3′ loxP sequences 3′ of a nucleic acid encoding the selectable marker, an MHC epitope 5′ to the 5′ loxP sequences or 3′ of the 3′ loxP sequence, an insertion end at the 5′ end of the transposable element, and an insertion end at the 3′ of the transposable element. A method is provided for detecting an antigenic epitope of a pathogen by infecting a pathogenic cell with a transposable element of the invention, wherein the infection results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell; transforming the pathogenic cell with a vector comprising a transposase; contacting a eukaryotic cell that can internalize the pathogenic cell with the pathogenic cell infected with the transposable element; contacting the eukaryotic cell with a specific binding partner that recognizes the MHC epitope; identifying the labeled eukaryotic cells and externalizing the bacteria cell. The externalized bacterial cell may be grown to produce a population of bacterial cells, and the nucleic acid sequence of the bacterial cell that has the integrated transposable element is identified. This nucleic acid sequence encodes the antigenic element of the panthogen. A method is also provided for generating a carrier vaccine by infecting a bacterial cell with the transposable element of the invention, wherein the transposable further comprises an antigen associated with a disease operably linked to the MHC epitope of the transposable element. The infection of the bacteria results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell. The pathogenic cell is then internalized into a eukaryotic cell and the eukaryotic cell is exposed to a specific bindned agent that recognizes the MHC epitope, identifying labeled eukaryotic cells are identified and lysed to externalized the bacteria cell, which is cultured to produce a population of bacterial cells. The nucleic acid sequence of the bacterial cell that has the integrated transposable element is identified, wherein the nucleic acid sequence encodes the antigenic element of the pathogen. The graving bacterial cell identified, and may be used as the carrier vaccine.

This is a § 371 U.S. national stage of PCT/US00/14687 filed May 26,2000, which was published in English under PCT Article 21(2), whichclaims the benefit of U.S. provisional application No. 60/136,210 filedMay 26, 1999.

FIELD

This invention relates to transposons, specifically to the use oftransposons to insert into a genome to identify antigenic epitopes. Thisinvention also relates to the identification of vaccine antigens.

BACKGROUND

The immune system is alerted to the presence of foreign infectiousagents by the presentation of complexes on the surface of the infectedcell. The complexes are composed of antigens derived from the pathogenand proteins of the Major Histocompatability Complex (MHC). Two separatepathways, MHC I and MHC II, drive cellular and humoral immune responses,respectively. In general, MHC I-presented antigens are derived fromcytoplasmic proteins. However in antigen presenting cells (APC), the MHCI-presented antigens are derived from an alternate pathway through alysosomal compartment (Morrison et al. J. Exp. Med 163:903-21, 1986;Pfeifer et al. Nature 361:359-62, 1993.) MHC II antigens are generallyderived from pinocytotic or phagocytic mechanisms (Morrison et al. J.Exp. Med 163:903-21, 1986).

Of the many pathogenic bacteria capable of mediating disease in humansand animals, intracellular pathogens present unique challenges inattempting to understand bacteria/host cell interactions. Intracellularpathogens are divided into two groups: those that reside within aphagolysosomal compartment (Salmonella sp, Mycobacterium tuberculosis,etc.) and those which reside within the cytoplasm (Listeriamonocytogenes, Shigella sp, etc.). Intracellular pathogens adapt totheir host cell environment by the selective secretion of proteinsdesigned to alter the normal structural and metabolic machinery of thehost cell, thus promoting bacterial survival and avoidance of hostimmune surveillance. Both phagolysosomal and cytoplasmic intracellularpathogens secrete proteins known to mediate their effects specificallywithin the host cell cytoplasm (Cornelis and Wolf-Watz, Mol. Microbiol.23:861-7, 1997; Collazo and Galan, Mol. Microbiol. 24:747-56, 1997; Fuand Galan, Mol. Microbiol. 27:359-68, 1998). Because cytoplasmiclocalization of the bacterial protein also infers access to thedegradative machinery of the host cell proteosome, these proteins werenamed Class I Accessible Proteins (CAPs).

Vaccination with Salmonella results in the production of a strongcellular and humoral response against the bacteria itself (Sztein etal., J Immunol 155:3987-93, 1995). However, the heterologous-antigenspecific immune response is variable and depends on several factors,including the nature of the antigen itself, the type of cell and tissuein which the antigen is expressed, the level of expressi n, and whetherthe antigen is presented and processed by the class I or class II MHCpathways. Results using either the SIV capsid antigen or the malariacircumspor zoite antigen, demonstrate that antigen-specific cytotoxic Tlymphocyte (CTL) responses are induced when the antigen is expressed inSalmonella (Flynn et al., Mol. Microbiol. 4:2111-8, 1990; Sadoff et al.,Science, 240:336-8, 1988; Valentine et al., Vaccine. 14:138-46, 1996).Other antigens have failed to elicit a CTL response even in similarexpression systems (Tite et al., Immunology 70(4):540-6 1990).

A plasmid containing a gene for a foreign antigen expressed from aeukaryotic promoter resulted in a strong cell-mediated response againstthe foreign antigen (Darji et al., Cell 91(6):765-75 1997); Schodel andCurtiss, Dev. Biol Stand 84:245-53, 1995).

A significant advance in the area of cancer vaccination has been theidentification of tumor-specific epitopes. In general, cancer vaccinesattempt to elicit an immune response to tumors by directingtumor-specific epitopes to various compartments of the immune system.Several strategies, which include vaccines composed of DNA, proteins,peptides, whole cells, carbohydrates and recombinant vectors, have beenused to generate tumor vaccines. The use of recombinant vectors includesthe use of live carrier vectors such as vaccinia, BCG, canarypox, andSalmonella, which are designed to stimulate the appropriate immuneresponses to tumors and infectious agents as a by-product of infection.Effective vaccines need to elicit strong, long-term, and multi-haplotypeprotection against a tenacious cancer. An ideal vaccine would satisfythese requirements and elicit an inescapable immune response bydelivering a wide-variety of antigens.

SUMMARY

A transposable element is provided that has a 3′ and a 5′ end. Thetransposable element includes a 5′ recombining site 5′ of a nucleic acidsequence encoding a selectable marker, a 3′ recombining site 3′ of thenucleic acid sequence encoding a selectable marker, a nucleic acidsequence encoding an MHC epitope 5′ to the 5′ recombining site or 3′ tothe 3′ recombining site, and an insertion end comprising an invertedrepeat sequence sufficient for integration of the transposable elementat the 5′ and the 3′ end of the transposable element.

In one embodiment, a transposable element is provided that has a 5′ anda 3′ end. The transposable element includes a 5′ loxP sequence 5′ of anucleic acid encoding a selectable marker, a 3′ loxP sequence 3′ of anucleic acid encoding the selectable marker, an MHC epitope 5′ to the 5′loxP sequences or 3′ of the 3′ loxP sequence, an insertion end at the 5′end of the transposable element, and an insertion end at the 3′ of thetransposable element

In another embodiment, a transposable element is provided that has a 5′and a 3′ end. The transposable element includes an antibiotic resistancecassette, a 5′ loxP sequence 5′ of the antibiotic resistance cassetteand a 3′ loxP sequence 3′ of the antibiotic resistance cassette, an MHCepitope 5′ to the 5′ loxP sequence or 3′ of the 3′ loxP sequence, anaffinity tag, an insertion end at the 5′ end of the transposableelement; and an insertion end at the 3′ of the transposable element.

In yet another embodiment a transposable element is provided that has a5′ and a 3′ end. The transposable element includes a kanamycinantibiotic resistance cassette, a loxP sequence comprising the sequenceshown in SEQ ID NO 11 located 5′ and 3′ to the antibiotic resistancecassette, a nucleic acid sequence encoding a transposase, a nucleic acidsequence encoding a MHC epitope, a nucleic acid sequence encoding a 6×histidine affinity tag, an insertion end at the 5′ end of thetransposable element; and an insertion end at the 3′ of the transposableelement.

Transposable elements have been engineered which can introduce in-frameinsertions throughout the chromosome of a bacterium. This system “tags”the gene and resulting protein, for use in identifying proteins secretedacross the membranes of the cell infected by the bacterium.

One particular embodiment of the method includes infecting a pathogeniccell with a transposable element of the invention, wherein the infectionresults in the integration of the transposable element in a nucleic acidsequence of the bacterial cell, transforming the pathogenic cell with avector comprising a transposase, contacting a eukaryotic cell that caninternalize the pathogenic cell with the pathogenic cell infected withthe transposable element, contacting the eukaryotic cell with a labeledantibody that recognizes the MHC epitope, identifying the labeledeukaryotic cells, lysing the labeled eukaryotic cells to externalize thebacteria cell, growing the externalized bacterial cell to produce apopulation of bacterial cells; and identifying the nucleic acid sequenceof the bacterial cell that has the integrated transposable element,wherein this nucleic acid sequence encodes the antigenic element of thepathogen.

In another embodiment, a method is provided for generating a carriervaccine. The method includes infecting a bacterial cell with thetransposable element of the invention, wherein the transposable elementfurther comprises an antigen associated with a disease operably linkedto the MHC epitope of the transposable element, wherein the infection ofthe bacteria results in the integration of the transposable element in anucleic acid sequence of the bacterial cell. The method also includescontacting a eukaryotic cell that can internalize the pathogenic cellwith the pathogenic cell infected with the transposable element,contacting the eukaryotic cell with a labeled antibody that recognizesthe MHC epitope, identifying the labeled eukaryotic cells, lysing thelabeled eukaryotic cells to externalize the bacteria cell, growing theexternalized bacterial cell to produce a population of bacterial cells;identifying the nucleic acid sequence of the bacterial cell that has theintegrated transposable element, wherein the nucleic acid sequenceencodes the antigenic element of the pathogen; and growing theidentified bacterial cell identified. The identified bacterial cell isthe carrier vaccine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the Tn5-DICE transposon.

FIG. 2 is a schematic representation showing the in-frame resolution ofTn5-DICE which was used to generate the expression of fusion proteinscontaining the SIINFEKL epitope and a 6×-histidine tag.

FIG. 3 is a schematic representation of some plasmids used for DICEanalysis. A. Pasmid carrying a Tn5-DICE resolvable transposon; B.Arabinose inducible cre recombinase plasmid pBAD33cre.

FIG. 4 is a schematic representation showing one embodiment of themethod developed to sequencing the Tn5-DICE-resolved CAPs. A. Suicideplasmid pAV353, containing a resolved copy of Tn5-DICE, was conjugatedinto a naladixic acid resistant, Cre expressing Tn5-DICE mutant B. Anampicillin and naladixic acid resistant transconjugant was obtained viaCre-loxP recombination. C. Isolated chromosomal DNA was restricted withEcoRI or SalI to clone 5′- or 3′-sequences flanking the originalSIINFEKL inaction, respectively.

FIG. 5 is a schematic representation showing the Tn5-HER2/neu/SOB(HER2/neu/String of Beads) construct.

FIG. 6 is a schematic representation showing the Tn5-HIV1/SOB construct.

FIG. 7 is a schematic representation of a DICE I transposome, which doesnot contain transposase, and can be used to identify CAPs presented bythe MHC I pathway.

FIG. 8 is a schematic representation of a DICE II transposome, whichdoes not contain transposase, and can be used to identify CAPs presentedby the MHC II pathway.

FIG. 9 is a schematic representation of a Salmonella-HER2/neu epitopecarrier vaccine.

FIG. 10 is a schematic representation of a Salmonella-HIV epitopecarrier vaccine.

FIG. 11 shows the Tn5 Mosaic end sequences.

FIG. 12 shows the DICE-I Resolved Sequence.

FIG. 13 shows the DICE-II Resolved Sequence.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO 1 is the nucleic acid sequence of a primer that can be used tosequence the gene in which a transposable element inserted.

SEQ ID NO 2is the nucleic acid sequence of a primer that can be used tosequence the gene in which a transposable element inserted.

SEQ ID NO 3 is the sequence of the O end.

SEQ ID NO 4 is the sequence of a mosaic end.

SEQ ID NO 5 is the sequence of an 1 end.

SEQ ID NO 6 is the SIINFEKL epitope.

SEQ ID NO 7 is the LLFGYPVYV epitope.

SEQ ID NO 8 is the ASFEAQGALANIAVDKA epitope.

SEQ ID NO 9 is the sequence of a 5′ PCR site, shown as position 54-77 ofFIG. 12.

SEQ ID NO 10 is the sequence of the 6× histidine, shown as position82-100 of FIG. 12.

SEQ ID NO 11 is the sequence of the loxP, shown a position 109-143 ofFIG. 12.

SEQ ID NO 12 is the sequence of the 3′ PCR site, shown as position145-167 of FIG. 12.

SEQ ID NO 13 is the sequence of a 5′ PCR site, shown as position 25-45of FIG. 13.

SEQ ID NO 14 is the sequence of the 5′ asparyginyl endopeptidasecleavage site, shown as position 34-45 of FIG. 13.

SEQ ID NO 15 is the sequence of the 3′ asparyginyl endopeptidasecleavage site, shown as position 97-108 of FIG. 13.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Transposable elements have been engineered which can introduce in-frameinsertions throughout the chromosome of a bacterium. This system “tags”the gene and resulting protein, allowing the identification of proteinssecreted across the membranes of the cell infected by the bacterium. Inone embodiment, the transposable elements contain an antibioticresistance cassette, two minimal loxP recombination sites, an MHC classI or class II epitope, and flanking insertion ends. A transposase, suchas the cre recombinase protein is expressed in trans from a plasmid, orcan be included in the transposable element. The cre recombinase loopsout the intervening sequences containing the antibiotic resistancecassette. When the transposable elements insert within a gene, theresolved insertion places the MHC class I or class II epitope in framewith the gene. Restriction sites allow the introduction of other markerproteins.

Certain embodiments of this technology, termed Disseminated Insertionsof Class-I Epitopes (DICE-I) (DICE-II for class II epitopes), allow therapid and accurate identification of proteins involved in bacterialpathogenesis. Uses for this technology include the identification ofvaccine and drug targets for therapy of a variety of bacteria pathogenicto humans and animals. In addition, this system can facilitate theassignment of function to genes previously identified through genomicanalysis. This method is also directly applicable to the generation ofhaplotype independent cytotoxic T lymphocyte (CTL) response to a givenantigen as a way of assessing patient immune response; measuring CTLresponse as a way of diagnosing specific infections; development ofhuman and animal vaccines that require a strong CTL response;identification of new bacterial carrier proteins that can be used togenerate a CTL response to infection; and augmentation of the immuneresponse by delivery of eukaryotic immune effectors.

The identification of CAPs secreted by the MHC class I or class IIpathway in response to host cell interactions are invaluable in thedesign of better bacterial carrier vaccines and to identify entire newclasses of potentially useful vaccine target proteins from differentpathogens and tumors, since CAPs possess unique access to the host'santigen processing and presentation machinery. In addition, because asubstantial proportion of open reading frames derived from whole genomeanalysis have no known function, a system which allows theidentification of CAPs secreted in response to host cell interactions,is an invaluable tool for understanding many levels of pathogen/hostcell interactions. Furthermore, CAPs represent useful vehicles for thedelivery of foreign epitopes by bacterial vaccine strains, such asSalmonella.

DICE-I and DICE-II have several inherent strengths in the identificationof CAPs. In some embodiments, DICE selection is conditional, host classI-accessible proteins are isolated as a consequence of being processedand presented in the context of H-2 K^(b), and host class I-accessibleproteins are isolated as a consequence of being processed and presentedin the context of I-A^(b). Moreover, only in-frame insertions, which donot alter secretory signals, are recovered. Selection can be made simpleand powerful, with interesting strains quickly recovered from a largepopulation of infected cells by flow cytometry. Since selection isspecific, bacteria cannot be recovered from macrophages that havepresented a MHC epitope from non-secreted intracellular proteins derivedby bacterial attrition within the phago-lysosome because these bacteriawould not be viable. Moreover, because the transposable elements cancarry an affinity tag such as 6×-hisitdine, the subcellular location ofthe protein can be visualized by microscopy, thereby enabling functionaland phenotypic inferences to be drawn about proteins with no knownhomology. Also, the protein can be readily assessed as an epitopecarrier by attenuating the strain and immunizing the appropriate animalmodel.

Abbreviations and Definitions

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. As used herein and in the appendedclaims, the singular forms “a” or “an” or “the” include plural referentsunless the context clearly indicates otherwise. Thus, for example,reference to “transposon” includes a plurality of such transposons andreference to “the antigen” includes reference to one or more antigensand equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

-   -   MOI multiplicity of infection    -   RT room temperature

Affinity Tag: A sequence which can be included in a transposable elementwhich can aid in the purification of the protein in which thetransposable element inserts. The term affinity tag refers to thenucleic acid sequence for the tag, and the tag protein sequence encodedby the nucleic acid sequence. Examples of affinity tags include, but arenot limited to: histidine, such as 6×histidine, S-tag,glutathione-S-transferase (GST) and streptavidin.

Animal: Living multicellular vertebrate organisms, a category whichincludes, for example, mammals, primates, and birds.

Antibiotic resistance cassette: A selectable marker that is a nucleicacid sequence which confers resistance to that antibiotic in a host cellin which the nucleic acid is translated. Examples of antibioticresistance cassettes include, but are not limited to kanamycin,ampicillin, tetracycline, chloramphenicol, neomycin, hygromycin, zeocin.

Cancer: Malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

CAPs: MHC Class I or Class II accessible proteins.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA may be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Deletion: The removal of a sequence of DNA, the regions on either sidebeing joined together.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Insertion Ends: Nucleic acid sequences that bind transposase. Ingeneral, insertion ends are 19 base pairs in length. In the constructsdescribed herein they are located 5′ (the 5′ insertion end) to the MHCepitope and 3′ (the 3′ insertion end) to the 3′ loxP sequence. Examplesof 5′ insertion ends include, but are not limited to, the I end of IS50R(e.g. SEQ ID NO:5, Genbank Accession No. U3299 1.1) and the mosaicsequence (SEQ ID NO:4, see Goryshin and Reznikoff Journal of BiologicalChemistry 273(13):7367-74). Examples of 3′ insertion ends include, butare not limited to, the O end of IS50R (e.g. SEQ ID NO:3, Genbankaccession No. U00004.1 and the mosaic sequence shown herein (see FIG.11).

IS50R: Insertion sequence (IS) type 50R. This IS element ends in shortinverted terminal repeats, designated the I and O ends (insertion ends)(e.g. see Genbank Accession Nos. U32991.1 and U00004.1, respectively).

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

loxP sequence: A target sequence recognized by the bacterial crerecombinase; loxP is the recombination site for the enzyme Crerecombinase. The loxP sequence was originally derived from bacteriophagePl (see Hoekstra et. Al., Proceedings of the National Academy ofSciences 88(12):5457-61 1991). In one embodiment, loxP sites are definedby the sequence ATAACTTCGTATAATGTATGCTA TACGAAGTTAT. A “minimal” loxPsequence is the minimal sequence recognized by the cre recombinase. Inone embodiment, minimal loxP sequence is as described in Hoekstra et.Al., Proceedings of the National Academy of Sciences 88(12):5457-611991. Specific, non-limiting examples include, but are not limited to,the sequence listed as Genbank accession No. MI0494.1. The 5′ and 3′loxP sequences must be identical. The loxP sites are represented by thesequence defined above to prevent premature transcriptional termination.

As used herein, these sequences are located upstream and downstream (5′and 3′, respectively) to a sequence encoding a selectable marker.

Mammal: This term includes both human and non-human mammals. Similarly,the terms “subject,” “patient,” and “individual” include human andveterinary subjects.

MHC Epitopes: Epitopes presented through the class I or class II MHCpathway, for which at least one antibody is available. The antibodybinds preferentially to the epitope complexed with MHC molecules, not tothe free epitope. Examples of class I MHC epitopes include, but are notlimited to the ovalbumin epitope, SIINFEKL (SEQ ID NO 6), and the HLA-A2restricted human T-cell epitope LLFGYPVYV (SEQ ID NO 7) from HTLV-1 (seeGenbank Accession No. B45714). Examples of class I MHC epitopes include,but are not limited to, the I-A^(b) restricted T-cell epitope,ASFEAQGALANIAVDKA (SEQ ID NO 8).

A MHC epitope “adjoins” a recombining site (i.e., a 5′ or 3′ recombiningsite) when the nucleic acid sequence encoding the MHC epitope is locatedeither 5′ of the 5′ recombining site of 3′ of the 3′ recombining site ina transposable element. Upon recombination of a transposable elementwith a genome, insertion a of the MHC epitope in the genome occurs, andthe MHC epitope is expressed along with a cellular protein. In oneembodiment, the MHC epitope is located within about 5000 bp of therecombining site. Alternatively, the MHC epitope can be located withinabout 1000 bp., 500 bp, 100 bp, 20 bp, 10 bp, or 0 by from therecombining site.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least 6 nucleotides, for example at least 15, 50, 100or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Ortholog: Two nucleotide sequences are orthologs of each other if theyshare a common ancestral sequence, and diverged when a species carryingthat ancestral sequence split into two species. Orthologous sequencesare also homologous sequences.

PCR: polymerase chain reaction. Describes a technique in which cycles ofdenaturation, annealing with primer, and then extension with DNApolymerase are used to amplify the number of copies of a target DNAsequence.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the DNA, RNA, and proteinsherein disclosed. Embodiments of the invention comprising medicamentscan be prepared with conventional pharmaceutically acceptable carriers,adjuvants and counterions as would be known to those of skill in theart.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, ethanol, sesameoil, combinations thereof, or the like, as a vehicle. The medium mayalso contain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The carrier andcomposition can be sterile, and the formulation suits the mode ofadministration. For solid compositions (e.g., powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, sodiumsaccharine, cellulose, magnesium carbonate, or magnesium stearate. Inaddition, to biologically-neutral carriers, pharmaceutical compositionsto be administered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides.

Probes and primers: Nucleic acid probes and primers may readily beprepared based on the amino acid sequences provided by this invention. Aprobe is an isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al., in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., inCurrent Protocols in Molecular Biology, Greene Publishing Associates andWiley-Intersciences (1987).

Primers are short nucleic acids, such as DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., in CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Intersciences (1987), and Innis et al., PCR Protocols. A Guide toMethods and Applications, 1990, Innis et al. (eds.), 21-27, AcademicPress, Inc., San Diego, Calif. PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of skill in the art willappreciate that the specificity of a particular probe or primerincreases with its length. Thus, for example, a primer comprising 20consecutive nucleotides of a cDNA or gene will anneal to a targetsequence such as a homolog of that gene contained within a cDNA orgenomic DNA library with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, in order to obtain greaterspecificity, probes and primers may be selected that comprise 20, 25,30, 35, 40, 50 or more consecutive nucleotides of the nucleic acidsequences herein disclosed.

The invention thus includes isolated nucleic acid molecules thatcomprise specified lengths of the disclosed gene sequences. Suchmolecules may comprise at least 20, 21, 25, 30, 35, 40, 50 or 100 ormore consecutive nucleotides of these sequences and may be obtained fromany region of the disclosed sequences. By way of example, the cDNA andgene sequences may be apportioned into halves or quarters based onsequence length, and the isolated nucleic acid molecules may be derivedfrom the first or second halves of the molecules, or any of the fourquarters. In particular, the DNA sequences may code for a unique portionof the protein, which has not been previously disclosed.

Purified: the term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein is more pure than the protein inits natural environment within a cell. In one embodiment, a preparationof a protein is purified such that the protein represents at least 50%of the total protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Recombining sites: Nucleic acid sequences that include invertedpalindromes separated by an asymmetric sequence at which a site-specificrecombination reaction can occur. In one specific, non-limiting example,a recombining site is a Lox P site (see above). In another specificnon-limiting example, a recombining site is a Flt sites. The FRTconsists of two inverted 13-base-pair (bp) repeats and an 8-bp spacerthat together comprise the minimal FRT site, plus an additional 13-bprepeat which may augment reactivity of the minimal substrate (e.g. seeU.S. Pat. No. 5,654,182). In other, specific non-limiting examples, arecombining site is a recombining site from a TN3, a mariner, or agamma/delta transposon.

Recombinase: A protein which catalyses recombination of recombiningsites (reviewed in Kilby et al., TIG, 9, 413-421 (1993); Landy, CurrentOpinion in Genetics and Development, 3, 699-707 (1993); Argos et al.,EMBO J., 5, 433-440 (1986)). One specific, non-limiting example of arecombinase is a Cre protein. Another specific, non-limiting example arecombinase is a Flp protein. Other specific, non-limiting examples of arecombinase are Tn3 recombinase, the recombinase of transposongamma/delta, and the recombinase from transposon mariner.

The Cre and Flp proteins belong to the lambda, integrase family of DNArecombinases. The Cre and Flp recombinases show striking similarities,both in terms of the types of reactions they carry out and in thestructure of their target sites and mechanism of recombination (see,e.g., Jayaram, TIBS, 19, 78-82 (1994); Lee et al., J. Biolog. Chem.,270, 4042-4052 (1995)). For instance, the recombination event isindependent of replication and exogenous energy sources such as ATP, andfunctions on both supercoiled and linear DNA templates.

The recombinases exert their effects by promoting recombination betweentwo of their recombining sites. In the case of Cre, the recombining siteis a Lox site, and in the case of Flp the recombining site is a Frt.Similar sites are found in transposon gamma/delta, TN3, and transposonmariner. These recombining sites are comprised of inverted palindromesseparated by an asymmetric sequence (see, e.g., Mack et al., NucleicAcids Research, 20,4451-4455 (1992); Hoess et al., Nucleic AcidsResearch, 14, 2287-2300 (1986); Kilby et al., supra). Recombinationbetween target sites arranged in parallel (i.e., s-called “directrepeats”) on the same linear DNA molecule results in excision of theintervening DNA sequence as a circular molecule. Recombination betweendirect repeats on a circular DNA molecule excises the intervening DNAand generates two circular molecules. Both the Cre/Lox and Flp/Frtrecombination systems have been used for a wide array of purposes suchas site-specific integration into plant, insect, bacterial, yeast andmammalian chromosomes has been reported (see, e.g., Sauer et al., Proc.Natl. Acad. Sci., 85, 5166-5170 (1988)). Positive and negativestrategies for selecting or screening recombinants have been developed(see, e.g., Sauer et al., J. Mol. Biol., 223, 911-928 (1992)). The useof the recombinant systems or components thereof in transgenic mice,plants and insects among others reveals that hosts express therecombinase genes with no apparent deleterious effects, thus confirmingthat the proteins are generally well-tolerated (see, e.g., Orbin et al.,Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)).

Sample: Includes biological samples containing genomic DNA, RNA, orprotein obtained from body cells, such as those present in peripheralblood, urine, saliva, tissue biopsy, surgical specimen, amniocentesissamples and autopsy material.

Selectable Marker: A polypeptide used to identify a cell of interestthat express the polypeptide. A selectable can be detected using anymethod known to one of skill in the art, including enzymatic assays,spectrophotometric assays, antibiotic resistance assays, and assaysutilizing antibodies (e.g. ELISA or immunohistochemistry). Specificnon-limiting examples of a selectable maker are luciferase, greenfluorescent protein (GFP), or beta-galactosidase. In one embodiment, aselectable marker is an enzyme. In another embodiment, a selectablemarker is an enzyme. In further embodiment, a selectable marker is anantigenic epitope. Specific, non-limiting examples of selectable markersof use are proteins that make a cell drug resistance (e.g. zeomycin,hygromycin, tetracycline, puromycin or bleomycin resistant).

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs of the nucleic acid and proteinsequences of the DICE transposons of the present invention will possessa relatively high degree of sequence identity when aligned usingstandard methods. This homology will be more significant when theorthologous proteins or cDNAs are derived from species which are moreclosely related, compared to species more distantly related.

Typically, homologs of the DICE transposomes of the present inventionare at least 50% identical at the nucleotide level and at least 50%identical at the amino acid level when comparing DICE transposomes ofthe present invention to a homologous DICE transposomes. Greater levelsof homology are also possible, for example at least 75%, 90%, 95% or 98%identical at the nucleotide level.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appt. Math 2:482, 1981; Needleman & Wunsch, J. Mot. Biol.48:443, 1970; Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444, 1988;Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al.Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al.,Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol.215:403-10, 1990, presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI web site.

Homologs of the disclosed DICE transposomes amino acid sequence maypossess at least 60% A, 70%, 80%, 90%, 95%, 98% or at least 99% sequenceidentity counted over full-length alignment with the amino acid sequenceof the disclosed DICE transposomes using the NCBI Blast 2.0, gappedblastp set to default parameters. Queries searched with the blastnprogram are filtered with DUST (Hancock, and Armstrong, 1994, Comput.Appl. Biosci. 10:67-70). Other programs use SEG.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequence will show increasingpercentage identities when assessed by this method, such as at least70%, 75%, 80%, 90%, 95%, 98%/, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs will typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence.

Alternatively, one may manually align the sequences and count the numberof identical amino acids in the original sequence and a referencesequence that is compared to the original sequence. This number ofidentical amino acids is divided by the total number of amino acids inthe reference sequence and multiplied by 100 to result in the percentidentity.

One of ordinary skill in the art will appreciate that these sequenceidentity ranges are provided for guidance only; it is entirely possiblethat strongly significant homologs could be obtained that fall outsideof the ranges provided. The present invention provides not only thepeptide homologs that are described above, but also nucleic acidmolecules that encode such homologs.

One indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the firs: nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions.

The present invention provides not only the peptide homologs that aredescribed above, but also nucleic acid molecules that encode suchhomologs.

Subject: Living multicellular vertebrate organisms, a category whichincludes, both human and veterinary subjects for example, mammals, birdsand primates.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transgenic Cell: Transformed cells which contain foreign, non-nativeDNA.

Transposable Element: Small, mobile DNA sequences that can replicate andinsert copies at random sites within a chromosome. In general atransposable element has nearly identical sequences at each end, andoppositely oriented (inverted) repeats. Naturally occurring transposableelements (transposons) code for the enzyme, transposase, that catalysestheir insertion. Bacteria have two types of transposons; simpletransposons that have only the genes needed for insertion, and complextransposons that contain genes in addition to those needed forinsertion. Eukaryotes contain two classes of mobile genetic elements;the first are like bacterial transposons in that DNA sequences movedirectly. The second class (retrotransposons) move by producing RNA thatis transcribed, by reverse transcriptase, into DNA which is theninserted at a new site.

The term “transposable element” includes transposons and transposomes.Using the method described herein, a transposable element can be used toidentify CAPs from the MHC class I or class II pathway.

Transposase: The enzyme responsible for transposition of transposons. Asused herein, refers to both the nucleic acid sequence (e.g., see GenbankAccession No. AAB60064, and the amino acid sequence (e.g. see GenbankAccession No. U 15573) Transposome: Mobile genetic element, which isable to transport itself to other locations within a genome. As usedherein, refers to a transposable element refers to a mobile geneticelement which does not contain transposase. Examples include, but arenot limited to DICE-I and DICE-II shown in FIGS. 7 and 8, respectively.

Transposon: A mobile genetic element, which is able to transport itselfto other locations within a genome. As used herein, refers to atransposable element containing transposase. Examples include, but arenot limited to Tn5-DICE shown in FIG. 2, Tn5-HER/neu/SOB shown in FIG. 5and Tn5-HIV1/SOB shown in FIG. 6.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art.

Variants of Amino Acid and Nucleic Acid Sequences: The production of thedisclosed DICE transposons can be accomplished in a variety of ways. Oneof ordinary skill in the art will appreciate that the DNA can be alteredin numerous ways without affecting the biological activity of theencoded protein. For example, PCR may be used to produce variations inthe DNA sequence which encodes the disclosed DICE transposomes. Suchvariants may be variants that are optimized for codon preference in ahost cell that is to be used to express the protein, or other sequencechanges that facilitate expression.

Two types of cDNA sequence variant may be produced. In the first type,the variation in the cDNA sequence is not manifested as a change in theamino acid sequence of the encoded polypeptide. These silent variationsare simply a reflection of the degeneracy of the genetic code. In thesecond type, the cDNA sequence variation does result in a change in theamino acid sequence of the encoded protein. In such cases, the variantcDNA sequence produces a variant polypeptide sequence. In order tooptimize preservation of the functional and immunologic identity of theencoded polypeptide, any such amino acid substitutions may beconservative. Conservative substitutions replace one amino acid withanother amino acid that is similar in size, hydrophobicity, etc. Suchsubstitutions generally are conservative when it is desired to finelymodulate the characteristics of the protein. Examples of amino acidswhich may be substituted for an original amino acid in a protein andwhich are regarded as conservative substitutions include: Ser for Ala;Lys for Arg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln;Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile orVal for Leu; Arg or Gin for Lys Leu or Ile for Met; Met, Leu or Tyr forPhe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ileor Leu for Val.

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, are minimized to enhance preservation ofthe functional and immunologic identity of the encoded protein. Theimmunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody to the disclosed DICEtransposomes; a variant that is recognized by such an antibody isimmunologically conserved. In particular embodiments, any cDNA sequencevariant will introduce no more than 20, for example fewer than 10 aminoacid substitutions into the encoded polypeptide. Variant amino acidsequences can, for example, be 80%, 90% or even 95% identical to thenative amino acid sequence.

Conserved residues in the same or similar proteins from differentspecies can also provide guidance about possible locations for makingsubstitutions in the sequence. A residue which is highly conservedacross several species is more likely to be important to the function ofthe protein than a residue that is less conserved across severalspecies.

Additional definitions of terms commonly used in molecular genetics canbe found in Benjamin Lewin, Genes V published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecuelar Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

EXAMPLE 1 Generation of Transposable Elements

Transposable elements have the ability to randomly distribute MHC classI or class II epitopes throughout a bacterial genome. Transposableelements are flanked at the 5′ and 3′ end with insertion ends, whichbind transposase. In general, insertion ends are about 19 nucleotides inlength. Examples of 5′ insertion ends include, but are not limited to,the I end of IS50R (see GenBank Accession No. U32991.1) and a mosaicsequence of the I and O end. Examples of 3′ insertion ends include, butare not limited to, the O end of IS50R (see SEQ ID NO U00004.1) and amosaic sequence of the I and the O end (see FIG. 10).

Transposable elements also contain a pair of recombining sites, such aspair of minimal loxP sequences, which upon interacting with arecombinase, such as a cre recombinase, allow the sequences locatedbetween the recombining sites to be removed upon insertion of thetransposable element into the bacterial genome. In one embodiment, the5′ loxP sequence is located 5′ to the nucleic acid sequence encoding aselectable marker and 3′ to the MHC class I or class II epitope. The 3′loxP sequence is located 5′ to the 3′ insertion end and 3′ to thenucleic acid sequence encoding a selectable marker. The loxP sequencesused in the present invention (SEQ ID NO 11) contain an example of aminimal sequence which allows loxP to retain its function. Longer loxPsequences may be used in the present invention. However, a longer loxPsequence will be inserted into the bacterial genome. Without being boundby theory, a smaller insertion into the protein is more likely to allowthe protein to function properly.

Transposable elements of the present invention also contain a nucleicacid sequence encoding a selectable marker, located between the loxPsequences, which allows for selection of bacteria containing thetransposable element plasmid. In one embodiment, the nucleic acidsequence encoding a selectable marker encodes antibiotic resistance. Thenucleic acid sequence encoding a selectable marker chosen may depend onthe bacteria into which the transposable elements are inserted. Forexample, if Salmonella is used, a kanamycin resistance cassette may beused in the transposable element. Examples of other antibioticresistance cassettes that may be used to practice the present inventioninclude, but is not limited to ampicillin tetracycline, chloramphenicol,neomycin, hygromycin, zeocin.

MHC class I or class II restricted epitopes are delivered to a bacterialgenome by the transposable elements of the present invention. The MHCepitope is located 3′ to the 5′ insertion end, and 5′ to the 5′ loxPsequence. The MHC epitope used has at least one antibody binding siteavailable. The antibody binds preferentially to the epitope complexedwith MHC molecules, not to the free epitope. Examples of class I MHCepitopes which can be used include, but are not limited to, theovalbumin epitope, SIINFEKL (SEQ ID NO 6), and the HLA-A2 restrictedhuman T-cell epitope LLFGYPVYV (GenBank Accession No. B45714) fromHTLV-1. Examples of class II MHC restricted epitopes which can be usedinclude, but are not limited to, the 1-A^(b) restricted T-cell epitope,ASFEAQGALANIAVDKA (GenBank Accession No. 228499).

Transposable elements of the present invention may also contain theTn5-transposase sequence. If present, transposase is located 3′ to thenucleic acid sequence encoding a selectable marker and 5′ to the 3′ loxP sequence. Upon addition of cre recombinase, the transposase andnucleic acid sequence encoding a selectable marker are removed.

The transposable elements of the present invention may be used totranspose MHC epitopes into the genome of a wide-variety of organisms,including bacteria. Examples of organisms that may be used to practicethe present invention include, but are not limited to Salmonella,Mycobacterium tuberculosis, Plasmodium, and Listeria monocytogenes.

EXAMPLE 2 Construction of Tn5-Based DICE Transposable Elements

A Tn5-DICE transposon was generated which consists of a Tn5-transposaseand an antibiotic resistance cassette (kanamycin) flanked at its 5′ and3′ ends by direct repeats of a minimal loxP recombination site (FIG. 1).The entire Tn5-DICE transposon is flanked by the IS50R I and O ends. The5′ end of the transposon consists of the Tn5 I-end, the H-2K^(b)restricted ovalbumin epitope SIINFEKL (SEQ ID NO 6), a 6×-histidine tag,and one loxP site, which are translationally in-frame. Tn5-DICE randomlydistributes the ovalbumin epitope, SIINFEKL (SEQ ID NO 6), throughoutthe bacterial chromosome. Epitope-tagged CAPs released from theinfecting bacteria are processed by the proteolytic machinery of thehost cell and the carried the ovalbumin epitope SIINFEKL (SEQ ID NO 6),is presented in the context of H-2K^(b) on the surface of the host cell(see FIG. 11). In this construct, the I end is amino acids 1-19, theSIINFEKL (SEQ ID NO:6) is at position 28-52, the 5′ PCR site is atposition 54-77, the 6×Histidine is position 82-100, the Lox P is atposition 109-143, the 3′ PCR end is at position 145-167, and the O endis at position 153-171 (see FIG. 12).

Tn5-DICE was constructed so that upon induction with Cre recombinase,the insertion is resolved at the loxP sites. The kanarnycin andTn5-transposase cassettes are segregated to non-replicating loops andlost. When the insertion is in-frame to a gene, the 49 amino acidresolved product creates a fusion protein carrying the SIINFEKL (SEQ IDNO 6) epitope (FIG. 2).

An E. coli donor strain (ATCC; 53323), containing both an F′ plasmid andthe Tn5-DICE bearing plasmid, pDE510 (tra-/mob) (FIG. 3A) was mated witha nalidixic acid-resistant Salmonella typhimurium strain (ATCC No.14028). The bacteria were mated by incubating together at a 1:1 ratio inLuria-Bertani broth at 37° C. for 12 hrs. Nalidixic acid and kanamycinresistant Salmonella transconjugants, which contain both the F′ plasmidand the Tn5-DICE bearing plasmid, pDES10(F′::Tn5-DICE), were isolated.The presence of F′::Tn5-DICE was confirmed using a P22 sensitivity test(Miller J. H. Experiments in Molecular Genetics Cold Spring HarborLaboratory Press (1972)) and by the ability to transfer the transposonkanamycin marker back into E. coli or Salmonella recipients at afrequency equal to F′ plasmid transfer frequencies. This is a controlexperiment to insure that the insertion is truly on the F′ plasmid. TheF′ plasmid transfers at a specific frequency. If the transposableelement is carried on the F′ plasmid, then it should be re-transferableto a new recipient at a rate equal to that of the initial mating.

The Salmonella-specific bacteriophage, P22 (HTint) (ATCC. 19585-B1), wasused to make a pooled (meaning that a donor culture of Salmonellastrains carrying the F′ plasmid with the transposon insertion is used tomake a phage lysate) lysate of the S. typhimurium transconjugantscarrying F′::Tn5-DICE. P22 transduction is a frequently used method oftransferring genetic markers between Salmonella strains. Because thereis no sequence homology to F′ in Salmonella, the P22 phage lysate wasused to mutagenize a second Salmonella recipient (ATCC 14028),Salmonella strain containing pBAD33cre. The lack of F′ homology in therecipient insured that kanamycin resistant transductants derived as aresult of transposition rather than homologous recombination.Transductants were selected by kanamycin resistance (30 μg/ml) on Luriaagar. The Cre recombinase in pBAD33cre (FIG. 3B) is under tightregulatory control of the pBAD promoter and mediates resolution and lossof the kanamycin resistance gene and the Tn5 transposase gene only whenthe strain is grown in the presence of arabinose (1 mM). The pBAD33creplasmid is unstable and is lost in 3-10 generations when Salmonellastrains bearing this plasmid are grown without selection.

The Salmonella-specific bacteriophage, P22 (HTint) was used to make apooled lysate of the S. typhimurium transconjugants carryingF′′::Tn5-DICE (a donor culture of Salmonella strains carrying the F′plasmid with the transposon insertion was used to make a phage lysate).Because there is no sequence homology to F′ in Salmonella, the P22 phagelysate was used to mutagenize a second Salmonella recipient, Salmonellastrain containing pBAD33cre, (ATCC 14028). The plasmid pBAD33cre wasconstructed as follows. Cre-recombinase was cloned by PCR amplificationfrom the cre-recombinase expressing Eschericia coli strain NS2114(Seifert, et al., Proc. Natl. Acad. Sci. 83:73540 (1986)). AClaI-HindeIII digest of the sub-cloned cre-recombinase gene was clonedinto a ClaI-HindeIII digest of the arabinose-inducible plasmid pBAD33(Guzman, et al., J. Bacteriol. 177(14):4121-30 (1995)). The lack of F′homology in the recipient insured that kanamycin resistant transductantsderived as a result of transposition rather than homologousrecombination. Transductants were selected by kanamycin resistance (30μg/ml) on Luria agar. The Cre recombinase in pBAD33cre (FIG. 3B) isunder tight regulatory control of the pBAD promoter and mediatesresolution and loss of the kanamycin resistance gene and the Tn5transposase gene only when the strain is grown in the presence ofarabinose (1 mM). The pBAD33cre plasmid is unstable and is lost in 3-10generations when Salmonella strains bearing this plasmid are grownwithout selection.

EXAMPLE 3 Identification of Strains Containing DICE Insertions

The pool of S. typhimurium mutants generated in EXAMPLE 1 was enrichedfor in-frame insertions of the resolved Tn5-DICE transposon within genesencoding secreted effector proteins by fluorescence activated cellsorting (FACS). If Tn5-DICE were randomly integrated, approximately ⅙(20,000) mutants of the 120.000 independent Tn5-DICE insertionsgenerated should contain resolved in-frame insertions. Of these, manyinsertions will be in metabolic genes that may be essential. Inaddition, many insertions will be in promoter or non-coding intergenicregions. Of the remaining mutants, far fewer will be contained withinCAPs. The precise number of CAPs in S. typhimurium is unknown. SinceDICE insertions within CAPs may be rare events, a sensitive selectionprocedure was required. With the appropriate cell marker, FACS enabledthe isolation of extremely rare mutants.

Infection of Macrophages

Femurs were harvested from 4-6 week old C57B1/6 mice (H-2 Kb). Bonemarrow cells were extracted by ravaging each end of the femur with a 3cc syringe containing a 30 gauge needle and 2 mls of RPMI. The bonemarrow cells were washed three-times with RPMI at 37° C. and resuspendedat 1×10′ cells/ml in RPMI 1640/10% EBS containing 20% L929 media as asource of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF).L929 media was derived by growing L929 cells (murine fibrosarcoma,American Type Culture Collection, Mannassas, Va.) and subsequentlyharvesting the media seven days after growing cells to confluence. Thecultures differentiated into bone marrow derived macrophages (BMDM) byculturing the bone marrow cells for six days at 37° C., 5% CO₂. BMDMwere resuspended in RPMI 1640/10% FBS and seeded into 6-well plates at1×10⁷ cells per well.

The pooled Tn-5 resolved SIINFEKL (SEQ ID NO 6) library (S. typhimurium)generated in EXAMPLE 1 was used to infect BMDM cells. An extensivelibrary of independent insertions of the Tn5-DICE transposon wasgenerated to insure that each gene encoded by S. typhimurium receivedmultiple “hits.” The pooled library was grown overnight in Luria broth(LB) at 37° C. with shaking. The pooled library was washed three-timesin RPMI 1640 and suspended in RPMI at 5×10⁸ cells/ml. The resuspendedlibrary (20 μl) was dispensed into individual wells containing adheredBMDM cells (MOI=1). A MOI of one or less limits multiple infectionswithin the same BMDM. A 1% infection rate is expected for S. typhimuriumin vitro. Cultures were centrifuged for two minutes at 200 rpm toinitiate contact and subsequently incubated at 37° C. for one hour. Thecultures were washed three times with 37° C. phosphate buffered saline(PBS, pH 7.4,9 g/l NaCl; 0.144 g/l KH₂PO₄; 0.795 g/l Na₂HPO₄). Thecultures were overlayed with three mls of RPMI 1640/10% FBS containing50 μg/ml gentamycin to kill extracellular bacteria, then incubated at37° C. for two hours. The cultures were washed three times with 37° C.PBS and the cells scraped from the plate, resuspended in 10 mls of RPMI1640/1% FBS, and incubated on ice.

FACS Analysis

The BMDM cells were incubated with FITC-conjugated anti-H-2 D^(b) andbiotinylated anti-H-2 K^(b)/SIINFEKL (5 μg 25-D1.2). TheH-2/K^(b)/SIINFEKL-specific antibody (25-D1.2) was available from R.Germain, National Institutes of Health. The I-Ab ASFEAQGALANIAVDKA-specific antibody (Y-ae) was a gift from Dr. Leszek Ignatowicz at theInstitute of Molecular Medicine and Genetics, Medical College ofGeorgia, Augusta, Ga.). Cells were labeled with antibody for 30 min at4° C. Anti-H-2 K^(b)/SIINFEKL, a monoclonal antibody only recognizes theSIINFEKL epitope (SEQ ID NO 6); Porgador, et al., Immunity 6(6): 715-26(1997)) when it is complexed with the class-I restrictive element H-2K^(b). Since neither BMDM cells nor wild-type Salmonella manufacturethis peptide, the infecting Salmonella strain containing the resolvedinsertion is the source of the (SEQ ID NO 6) epitope. Cells were washedthree-times in 4° C. PBS and incubated with one μg phycoerythrin (PE)conjugated streptavidin (Caltag).

FACS analysis was used to identify and isolate Salmonella-infectedmacrophages that contained in-frame resolved transposon insertionswithin genes having access to the class-I antigen processing andpresentation pathway of the macrophage. BMDM infected with theSalmonella-DICE library were sorted by first gating on the forward andside scatter population characteristic for macrophages. Bright red(PE-anti-H-2 K^(b)/SIINFEKL) and bright green (FITC-conjugated anti-H-2D^(b)) populations, visualized in the double positive quadrant, weresorted into a five ml polypropylene tube containing two mls of RPMI1640/1% FBS. The sorted cells were centrifuged, lysed in LB/1% TritonX-100, then plated on LB agar and incubated at 37° C. overnight torecover Salmonella-DICE strains.

Infected BMDMs lacking CAP insertions can be recovered as a consequenceof aggregate formation in the flow sorted population. To ensure thatrecovery was due to phenotypic expression of H-2 Kb/SIINFEKL, therecovered bacterial colonies were counted, pooled, and subjected to twoadditional rounds of FACS sorting to enrich for Salmonella mutantscontaining CAP insertions. Individual isolates were subjected to anadditional round of FACS analysis to confirm their phenotype. Salmonellainfecting the double positive BMDM were removed and grown forconfirmation and sequencing.

EXAMPLE 4 Sequencing of CAP Genes

To determine the identity of CAPs containing in-frame SIINFEKL (SEQ IDNO 6) insertions, a unique system allowing specific and efficientidentification of CAP genes was developed (FIG. 4). The system was alsoused to efficiently retransduce Tn5-DICE mutants and reconfirm theirphenotypes. A KpnI-SacI fragment of a plasmid carrying the resolvedTn5-DICE transposon (pAV353a) was cloned into an ampicillin-resistantsuicide vector, pGP704, to yield plasmid pAV353 (FIG. 4A).

Plasmid pAV353 (amp′ tra⁺ mob*) was transformed into E. coli S17 λpir(Kinder, S. A. et al. Gene 136, 271-5 (1993)an ampicillin resistant,nalidixic acid sensitive donor strain, and conjugated into spontaneousnaladixic acid resistant, Cre expressing S. typhimurium Tn5-DICE mutantCAP mutants containing pBAD33cre. Transconjugants (amp′ nal′) carryingan integrated copy of plasmid pAV353 at the chromosomal loxP site wereselected following induction of the Cre recombinase, by selectingnaladixic acid and ampicillin resistant transconjugants (FIG. 4B).

Chromosomal DNA was isolated, digested for 2 hours at 37° C. with one ofseveral possible restriction endonucleases (see FIG. 4A), to allowcloning of either 5′- or 3′-DNA sequences flanking the original SIINFEKL(SEQ ID NO 6) insertion. Digested DNA was absorbed over a DNApurification column to remove the restriction endonuclease, and ligatedovernight at 15° C. Ampicillin resistant transformants were furtheranalyzed using Tn5-DICE specific primers 5′GCGGATATCCACCACCACCACC-3′(ClaI, SalI, XhoI, or KpnI digests) or 5′-TATGCCCGGGCCGTGGTGGTGG-3′(EcoRI, SacI digests).

Upon transformation into E. coli S17λpir, re-ligated circular fragmentscontaining pAV353 form functional replicons resulting inampicillin-resistant transformants. Re-ligated chromosomal fragmentscarrying the integrated plasmid pAV353 form functional replicons in E.coli S17 λpir and carry either 3′-(i.e. SalI) or 5′-(i.e. EcoRI)sequences flanking the original SIINFEKL (SEQ ID NO 6) insertion (FIG.4C). Ampicillin-resistant transformants were further analyzed usingTn5-DICE specific primers 5′-GCGGATATCCACCACCACCACC-3′ (ClaI, SalI,XhoI, or KpnI digests) (SEQ ID NO 1) or 5′-TATGCCCGGGCCGTGGTGGTGG-3′(EcoRI, SacI digests) (SEQ ID NO 2).

As shown in Table 1, the Tn5-DICE transposon (FIG. 2) enabled theidentification of class-I-MHC-accessible S. typhimurium proteins in bothmacrophages and an intestinal epithelium cell line (see EXAMPLE 4). S.typhimurium proteins not predicted to reach the class I pathway of thehost cell were identified. In addition, in at least one instance, abacterial effector protein unique to S. typhimurium secreted into thecytoplasm of the host cell has been identified (LS28). Characterizationof the immune response to each CAP identified may enable theconstruction of highly specific carrier vaccines, allowing immuneresponses to be tailored to the life cycle of specific pathogens.

TABLE 1 Salmonella genes identified by DICE Function/ Strain Gene Comp.*Bacteria CMT-93 Homology LS28 ams** S S. t., S. typhi + Protease IVSIIN16 argT P S. t., S. typhi, − Arginine E. c. Transport SIIN17 fhuA PS. t., S. typhi, − Iron E. c Transport SIIN27 S2OMP OMP S. t., S. typhi,− Outer E. c, K. p. Membrane Protein SIIN15 htpG S? S. t., S. typhi, +High E. c Temperature Heat Shock Protein SIIN29 hemK C S. t., S. typhi,− Heme E. c Biosynthesis SIIN50 ims75 S S. t., S. typhi + impairedmacrophage survival, MIP SIIN61 ORF S S. t., S. typhi + Unknown SIIN71hemL C S. t., S. typhi, − Heme E. c Biosynthesis S. t. = Salmonellatyphimurium; S. typhi = Salmonella typhi; E. c. = E. coli

EXAMPLE 5 Confirmation of the DICE Method

To confirm the validity of the DICE screen, several studies wereperformed to insure that the CAP epitope identified was present on thesurface of the antigen presenting cell (APC) and that mutants were ableto stimulate T-cell specific immunity. In addition, the route of antigendelivery was investigated to determine if proteins delivered by DICEmutants were accessible to the class-I MHC pathway by an alternateantigen processing and presentation pathway or directly into theendogenous pathway by translocation across the phago-lysosomal barrier.

Fluorescence Microscopy

The Salmonella DICE strain LS28 was transfected with a plasmid whichconstitutively expresses green fluorescence protein (GFP). This strain(LS28GFP) of a resolved S. typhimurium/SIINFEKL was used to infect H-2K^(b) restricted BMDM in vitro and then fluorescently labeled using themonoclonal antibody 25-D1.2 using the methods described in EXAMPLE 2.The infected BMDM cells were imaged using wide field fluorescenceimaging. H-2 K^(b)/SIINFEKL complexes were observed on the cell surface,demonstrating that BMDM derived the SIINFEKL epitope (SEQ ID NO 6) fromLS28GFP.

To examine the route of antigen processing, several of the isolated DICEmutants were used to infect the H-2K^(b) restricted murine intestinalepithelial line CMT-93 (ATCC, Manassas, Va. catalog number CCL-223).CMT-93 cannot present antigen delivered by Salmonella when the ovalbuminepitope is expressed intracellularly within the bacteria, suggestingthat CMT-93 cells do not contain an alternate antigen processingpathway. The most likely explanation for CMT-93 presentation of SIINFEKL(SEQ ID NO 6) on its cell surface is that the epitope was delivereddirectly to the endogenous pathway as a fusion with a type III secretedprotein.

The H-2 K^(b)/SIINFEKL specific CD8⁺ T-cell hybridoma B3Z (Karttunen, etal., Proceedings of the National Academy of Sciences 89:6020-24 (1992))is a reporter cell which turns blue when it encounters its ligand. B3Zwas used as an indicator of the presence of the H-2 K^(b)/SIINFEKLcomplex on the surface of CMT-93. The presence of blue B3Z cellsindicates that the H-2 K^(b)/SIINFEKL complex is recognized by aspecific T-cell receptor and is delivered by a bacterial protein.

Monolayers of CMT-93 cells (3×10⁴ cells/well) were infected with LS28 atan MOI of 1 in a 96 well tissue culture plate. Cultures were incubatedat 37° C. for one hour, washed of non-invasive Salmonella, and overlayedwith fresh media containing gentamycin (50 μg/ml). The cultures wereoverlayed with 3×10⁴ cells/well of B3Z cells and centrifuged to initiatecell-to-cell contact. The cultures were incubated at 37° C. for sixhours, then each well was washed, fixed (2% formaldehyde/0.2%glutaraldehyde) and incubated in developing buffer (1 mg/ml X-gal; 5 mMK³Fe(CN)₆; 5 mM K₄Fe(CN), 3H₂O; 2 mM MgCl₂). The cells were imaged usinglight microscopy. The presence of blue B3Z cells indicates that theSIINFEKL epitope is being targeted directly to the cytoplasm of the hostcell. This data is significant because it indicates that Salmonella isusing a translocation apparatus to target these proteins into thecytoplasm. These data indicate that access to the class-I MHC pathway bySalmonella is cell type dependent.

To confirm that the stimulation of B3Z was specific (stimulation ofT-cell specific immunity only when B3Z encounters CMT-93 cells infectedwith Salmonella), similar experiments were performed using wide-fieldfluorescence microscopy to visualize the CMT-93:B3Z interaction. CMT-93cells (2×10⁵/well, chambered coverglass #1.5) were infected with LS28GFP(37° C., 1 hour, MOI=10), overlayed with media containing gentamycin (50μg/ml). The cultures were seeded with B3Z T-cell hybridomas (2×10⁵cells) and incubated at 37° C. for 12 hours. The cultures were washedthree-times with PBS, fluorescent stained for cell membranes (TMA-DPH,Molecular Probes) and β-galactosidase (C₁₂FDG, Molecular Probes), andvisualized on an Advanced Precision Instruments deconvolutionmicroscope. Stimulation of B3Z was due to cognate interaction of B3Zwith infected CMT-93. The results provide visual evidence of bacterialprotein translocation. These results demonstrate that DICE analysis canbe used to isolate proteins having direct access to the class-I MHCpathway of the host cell, which is cell type specific. In addition, DICEstrains stimulate a specific T-cell response, due to the presence of theDICE strain.

β-galactosidase Assay

The ability of infected cells to present antigen to a T-cell reporterwas also assayed using a β-galactosidase assay. The T-cell reporter is aT-cell hybridoma (a fusion between a T-cell and a tumor cell) thatrecognizes the SIINFEKL epitope when presented in the context of theclass I MHC allele H-2K^(b). When the T-cell encounters the SIINFEKL/H-2K^(b) complex, it initiates synthesis of β-galactosidase. When incubatedin the presence of a substrate (X-gal), the cell turns blue. The cellwill turn blue only if this specific interaction has occurred. H-2K^(b)-restricted epithelial cells (ATCC No. CCL-223) were infected withseveral Salmonella strains isolated by flow cytometric analysis usingthe methods described above in EXAMPLE 2. The cells were then infectedwith 1×10⁷ CFU (MOI=100) of each of several DICE mutants isolated as inEXAMPLE 2. After 1 hr at 37° C., the wells were washed 3× with phosphatebuffered saline (PBS) and overlayed with 1×10⁵ B3Z cells. Cell to cellcontact was initiated by centrifuging at 200×g for 2 minutes. Thecultures were incubated at 37° C./5% CO₂ for 6 hrs. The cells werewashed 3× with PBS and fixed in a solution of PBS containing 1%formaldehyde and 0.2% glutaraldehyde for 5 minutes at 4° C. The cellswere then overlayed with a solution of PBS containing 1 mg/ml X-gal, 5mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl₂.The cells were allowed to incubate at 37° C. overnight and examinedmicroscopically for the presence of blue cells.

Several of the Salmonella clones turned blue, demonstrating that theSIINFEKL epitope is actively directed across the phagolysosomal borderby Salmonella after infection and is processed into the class I MHCpathway.

EXAMPLE 6 In Vivo T-cell Immunity

Access to the endogenous pathway of the host cell infers access to theclass-I MHC processing and presentation pathway of the host. Vaccinesthat carry antigens within CAPs should be able to stimulateantigen-specific cell-mediated immune responses. In vivo T-cell immunityto these antigens is the best measure of the ability of these vaccinesto stimulate appropriate responses.

C57B1/6 mice were orally immunized with several DICE strains. Briefly,female C57B1/6 mice (6-8 weeks old) were immunized by oral gavage with1×10 ⁷ CFU of each Salmonella DICE mutant.

The ability of these strains to stimulate T-cell responses in vivo canbe assessed by traditional CTL assays, H-2 K^(b)/SIINFEKL tetrameranalysis (using K^(b)/SIINFEKL tetramers obtained from the NIH AIDSReagent Program), and tumor protection assays. This combination ofmeasurements of T-cell immunity is used to confirm both the stimulationof antigen-specific T-cell populations and whether these T-cells arefunctional. The H-2K^(b)/SIINFEKL tetramers provide an extremelysensitive method of assessing the effect of vaccination upon the T-cellpopulation in the immunized animal. A positive effect would bemanifested by an increase in total antigen-specific T-cells afterimmunization. An increase in antigen-specific T-cells followingimmunization however tells little about the functionality of thesecells. If you have stimulated an antigen-specific population byimmunization, the vaccine would be poorly constructed if the stimulatedcells could not kill their targets. The CTL assays provide a necessaryand accurate measure of the functionality of the antigen-specific T-cellpopulation. Tumor cells which express the SIINFEKL epitope are used astargets for the assay. If the vaccine stimulates an antigen-specificT-cell population and these cells are able to efficiently kill theirtargets, then the vaccine can be considered to effectively engender aprotective immune response.

EXAMPLE 7 Construction of a Heterologous Antigen Breast Cancer Vaccine

The transposable elements of the present invention allow for rapididentification of CAPs, which may serve as beneficial targets forvaccine development. Since CAPs have access to the host immune system,vaccines against viruses, bacteria, and cancer can be constructed usingCAPs as vaccine carriers that target protective epitopes (for examplepieces of proteins from foreign infectious agents or cancer cells)directly to the cytoplasmic compartment of APCs. Access to the class Ior class II pathway of the host cell indicates that many proteins mayserve as attractive vaccine targets. Heterologous antigen expressed bySalmonella vaccine strains may induce a protective immune response inanimals and humans. Heterologous antigen-specific immunity can consistof both local and systemic Th1 or Th2 type immune responses.

HLA-A2-restricted epitopes derived from HER2/neu deposited directly intothe cytoplasmic compartment of the APC by CAPs may result in better MHCclass I presentation, thus greatly enhancing induction of cell-mediatedimmunity. Her2/neu is an epidermal growth factor-like protein whoseupregulation is associated with a variety of cancers of the breast andother tissues. Engendering a strong and persistent cellular immuneresponse is essential for protective immunity to tumors such asHER2/neu-elevated breast cancer. This example describes the constructionand delivery of HER2/neu/SOB (HER2/neu/String of Beads) insertionsthroughout the chromosome of S. typhimurium, using a variant of theTn5-DICE transposon shown in FIG. 2.

HER2/neu/SOB Library Construction

A transposon system was developed to generate a library of epitopeinsertions containing the HLA-A2-restricted HER2/neu epitopes (FIG. 5).HER2/neu/SOB carries a 6×-histidine site, the HLA-A2-restricted HTLV-1tax epitope LLFGYPVYV and three HLA-A2 restricted HER2/neu epitopesHER2/neu₍₃₆₉₋₃₇₇₎, HER2/neu₍₇₇₃₋₇₈₂₎, and HER2/neu₍₆₅₄₋₆₆₂₎. Resolvedin-frame insertions of Tn5-HER2/neu/SOB creates an 81 amino acid productencoding each epitope.

Initially, wild-type S. typhimurium (strain 14028s) is used to avoidpossible interaction between the attenuating mutation and the DICEinsertions. The DICE insertion in the strains that present antigen bestare transduced into, for example, three other strain backgrounds to testtheir immune response in HLA-A2.1 transgenic mice. Attenuated strainswill contain mutations in aroA. AroA is an enzyme involved in thebiosynthetic pathway for Aromatic amino acids. Mutations in the aroAlocus severely attenuate Salmonella vaccine strains thereby diminishingthe ability of the vaccine strain to disseminate and cause disease.Alternatively, attenuated strains CL401 or CL553 can be used (twoSalmonella typhimurium strains shown in our lab to be severelyattenuated for virulence. The location of the mutations are unknown).aroA can be used because mutations in aromatic amino acid biosynthesisare used in CV908 (a Salmonella typhi vaccine strain) that appears to beone of the best S. typhi vaccines.

Using the methods described above in EXAMPLES 1-3, P22, is used to makea lysate of the Salmonella strain containing F′::HER2/neu/SOB. The poolof S. typhimurium mutants are enriched for in-frame insertions of theHER2/neu/SOB cassette within CAPs by FACS as described above, withmodifications noted below.

Identification of Salmonella Isolates Able to Facilitate HTLVItax ClassI Presentation

Salmonella SOB-containing proteins that direct peptides into the class Ipathway from a library of Salmonella strains which contain the SOBpeptide can be identified using a monoclonal antibody specific toHTLVItax/A2.1.

BMDM isolated from H-2K_(b)/HLA-A2* transgenic mice (C57B1/6 background)are seeded onto 6-well tissue culture plates (1×10⁷ cells/well) andinfected with the pooled S. typhimurium/HIV-1/SOB library (37° C.,MOI=10). After one hour, the cells are washed, overlayed with RPMI1640/10% FBS (gentamycin, 50 μg/ml), and incubated for 2 hours (37° C.).The cells are harvested, washed, and suspended in 10 ml RPMI 1640/1%FBS. The BMDM are labeled with FITC-conjugated anti-H-2 D^(b) (Caltag)and biotinylated A6-TCR chimeric antibodies (a chimeric antibody whichrecognizes HLA-A2 complexed with the HTLV-Itax epitope LLFGYPVYV(obtained from Dr. Jonathan Schneck, Johns Hopkins University; O'Herrin,et.al., Journal of Experimental Medicine 186(8): 1333-45)to tag classI-expressing cells presenting the ta peptide in the context of HLA-A2.The biotinylated A6-TCR is subsequently labeled with PE-streptavidin(Caltag). The BMDM are re-suspended in RPMI 1640/1% FBS (4° C.) andsorted using FACS analysis by gating on populations expressing both H-2D^(b) and A6-TCR Bacteria recovered from the sorted cells, are pelletedand lysed in LB broth containing 1% triton X-100 followed by plating onLB-agar as described above in EXAMPLE 2.

To identify the gene carrying the in-frame HER2/neu/SOB insertion, a DNAtemplate for sequence analysis is generated using a variation of theTAIL PCR method (see EXAMPLE 3). This method employs the use ofsequential, tandem oligonucleotides that prime within the resolvedHER2/neu/SOB insertion and amplifies epitope insertions thus identifyingthe region flanking the insertion. The entire cycling procedure isperformed sequentially as a series of primary, secondary and tertiaryreaction conditions. The primary and secondary conditions are performedin volumes of 100 μl. All cycling conditions are as published (Liu andWhittier, 1995). Briefly, This method utilizes a complex array ofmelting and annealing procedures to determine the sequence flanking theinsertion. To accomplish this, three tandem primers are used to “walk”down the insertion and amplify from a fourth random primer. Afteramplification, the fragment is gel purified and cloned into a sequencingvector.

Alternatively, chromosomal preparations are made from each isolatedbacterium containing a CAP insertion. 1 μg of the chromosome is digestedwith the restriction enzyme pstI for 1 hr at 37° C. and then purified.The chromosome is then ligated by the addition of ligase overnight at15° C. The circularized mix is then subjected to inverse PCR using theprimers CTACTAGTATGGATGGTGTC and CTAGAACCAGAT GTGTATAAG. The PCR mix isas follows: 1 mM each primer, 10 ng template, 0.2 mM dNTP (dATP, dCTP,dGTP, dTTP), 0.5 Taq polymerase, 10 μl 10×PCR buffer, 1 mM MgCl₂, andH₂O to 100 μl. Cycling conditions are melting: 95° C., 30s; annealing:55° C., 1 min; extension: 72° C., 3 min; 35 cycles.

EXAMPLE 8 Characterization or Heterologous Antigen Breast CancerVaccines

After the construction of the S. typhimurium/HER2/neu vaccine straindescribed in EXAMPLE 7, the ability of the vaccine to induceepitope-specific, cell-mediated immune responses in HLA-A2 transgenicmice is characterized and quantified. An advantage of the Salmonellavaccine system is that there is a relevant small animal model, themouse, in which to evaluate the safety and efficacy of the vaccineconstructs developed.

Vaccine candidates can be chosen based upon criteria such as: 1) genesencoding CAPs proteins must be conserved between S. typhimurium and Styphi (as judged from the Salmonella genome projects); and 2) CAPscarrying epitope insertions must be recoverable upon repeatedindependent flow sorts. In previous experiments, all strains containinggene insertions that resulted in presentation of SIINFEKL in H-2K^(b)were recovered a second time. Other criteria may also be used.

S. typhimurium HER2/neu/SOB vaccine strains isolated in EXAMPLE 7 areused to orally immunize K^(b)/HLA-A2 transgenic mice. The mice wereimmunized by oral gavage with 1×10⁷ infectious units of Salmonella. Theresponse generated from each vaccine is subsequently analyzed using thefollowing methods.

HLA-Tetramer Construction and Analysis of Epitope-Specific T-Cells

The T-cell response to vaccination may, for example, be initiallyassessed by HLA-tetramer analysis of T-cell populations derived from thespleens and mesenteric lymph nodes of vaccinated, sham vaccinated, andunvaccinated HLA-A2 transgenic mice. The HLA-A2 transgenic mice utilizedwere from Dr. Linda Sherman, Scripps Institute, LaJolla, Calif. Toassess the class I response to each HER2/neu epitope, HLA-A2 tetramerscontaining each HER2/neu epitope plus one irrelevant control are used.HLA-A2 and β2-microglobulin expressing plasmids can be obtained from Dr.John Altman, Emory University. Conversely, tetramers are available fromthe Aids Reagent Repository at the National Institutes of Health,Bethesda Md. Freshly isolated spleen cells and cells derived frommesenteric lymph nodes from K^(b)/HLA-A2 mice immunized with each S.typhimurium HER2/neu/SOB vaccine are labeled with FITC-conjugatedanti-CD8 and each respective PE labeled HLA-A2 tetramer. Specifically,1×106 cells are labeled with 1 μg of each respective tetramer and 5 μgof anti-CD8 antibody for 30 min at 4° C. The effector status of thetetramer positive CD8 populations are further characterized by assessingthe level of expression of CD28, CD44, and CD62. These markers arehallmarks of the state of differentiation of the effector cells. Eachcell population will be labeled with 1 μg of each respective marker for30 min at 4° C. The CD8⁺/CD44lo/CD62⁺ phenotype correlates with a memorypopulation of splenic effector cells. From this data, the nature of thecellular response to vaccination is characterized. Ideal vaccinecandidates should yield a strong memory CTL population that is capableof rapid upregulation after restimulation by the infectious agent thevaccine was designed for.

CTL Lysis of HIV Epitope Expressing Targets

Epitope-specific T-cells resulting from vaccination of K^(b)/HLA-A2transgenic mice with tumor epitopes are capable of mediating killing ofhuman HLA-A2⁺ tumor targets. To assess the ability of Salmonella vaccinestrains to generate HER/neu-specific CTLs, assays designed to measurethe lytic capacity of the CTL population generated as a result ofvaccination can be used. To measure specific immune response toHER2/neu, a chromium release assay can be used to measure the ability ofCTLs generated in vaccinated mice to kill HER2/neu elevated, HLA-A2′tumor targets derived from the Oregon Health Sciences University tumorbank. The chromium release assay is a standard method used for thedetermination of the ability of activated cytotoxic T-cells to killtheir targets. Briefly, target cells are loaded with CTS, washed, andincubated with T-cells at effector to target ratios ranging from 1:1 to1:10,000. Killing is a measure of the amount of radioactive chromiumreleased into the culture supernatent at various times after incubation.Spleens from naive and infected animals are removed from mice 14-49 dayspost infection, and splenic cells collected for tetramer analysis andCTL assays. Secondary stimulation may be necessary before a CTL responseis observed. T1 (HLA-A2⁺−H-2^(d)) target cells loaded with either anindividual HLA-A2-restricted HER2/neu epitope or one irrelevant epitopecan be used. T1-cells are good secondary stimulators because theyexpress large amounts of HLA-A2 and can be easily loaded withHLA-A2-restricted epitopes. Alternative methods of stimulation includeincubation with Concanavalin A or through the T cell receptor usinganti-CD3 antibodies. Concanavalin A is a plant mitogen that broadlystimulates T-cells. Anti-CD3 similarly stimulates T-cells my mimickinginteraction with an antigen presenting cell. HLA-A2 tetramer positiveT-cell clones for each individual epitope can be isolated and preserved.

EXAMPLE 9 Construction of a Heterologous Antigen HIV Vaccine

An HIV-1 vaccine was constructed using a modified version of Tn5-DICE .As shown in FIG. 6, the vaccine, Tn5-HIV1/SOB (human immunodeficiencyvirus 1/string of beads) carries a 6×-histidine site, theHLA-A2-restricted HTLV-1 tar epitope, and five HLA-A2-restricted HIV-1epitopes (p 17₇₇₋₈₅; p24₁₉₃₋₂₀₃; RT₂₆₇₋₂₇₇; gp160₃₁₃₋₃₂₂; and nef₇₁₋₈₀).Resolved in-frame insertions of Tn5-HIV1/SOB create a 109 amino acidproduct encoding each epitope.

The Tn5-HIV 1/SOB construct was transferred to a Nal′ Salmonellarecipient by conjugation, and P22 was used to make a pooled lysate,using the methods described in EXAMPLES 1 and 6. Phage lysates were usedto mutagenize S. typhimurium (wild-type strain 14028s) and S. typhi(Ty21 a vaccine strain). Salmonella molecules which elicit appropriateCTL responses are selected and tested further for their ability toengender protective immune responses. Two measures of effectiveness maybe considered in assessing the efficacy of these vaccines. First, arethe vaccines able to elicit a protective response against cellsexpressing the epitopes? Second, do these vaccines elicit a protectiveresponse against a viral challenge? Variations on the methods outlinedabove will be used to assess the efficacy of the vaccine both in vitroand in vivo.

EXAMPLE 10 Construction of DICE-I and DICE-II Transposomes

The Tn5-DICE transposon shown in FIG. 2 can be engineered to accept avariety of different elements. For example, transposomes which can beused to identify Salmonella proteins (or those from a variety ofinfectious bacterial agents described above) which cycle into the MHCclass I or class 11 (MHC, HLA) pathway can be generated. Examples oftransposomes which can be used to identify Salmonella proteins whichcycle into the MHC class I or class II pathway include DICE I (FIG. 7)and DICE II (FIG. 8), respectively. The original Tn5-based DICEtransposon was modified in DICE-I and DICE-II by removing thetransposase. Removal of transposase provides many advantages. Itstabilizes the insertion, improves the efficiency of libraryconstruction because many steps in the process are eliminated, forexample the mating step. Removal of the transposase also increases therange of bacteria in which transposons can be used. Incorporation of thetransposome into the chromosome of the bacterium can be performed by asimple electroporation procedure. The transposomes shown in FIGS. 7 and8 have also have excessive secondary structure remove. These secondarystructures, present in Tn5-DICE, made PCR and cloning lessstraightforward. Unique 5′ and 3′ PCR primer sites have been added tofacilitate inverse PCR. The I- and O-ends were changed to mosaicsequences to enable efficient transposome construction.

DICE-I contains the ovalbumin epitope SIINFEKL to identify bacterialproteins which cycle into the MHC class I pathway. However, other MHCclass I epitopes can be used, for example the HTLV-1tax epitopeLLFGYPVYV (SEQ ID NO: 7), as well as other epitopes known in the art.

DICE II contains an I-A^(b) restricted T-cell epitope, ASFEAQGALANIAVDKA(SEQ ID NO 8). However, other MHC class II restricted epitopes can beused, including the anti-I-A/^(k)/Hen Egg Lysozyme (HEL₄₆₋₆₁) or theanti-I-A^(k)/Hen Egg Lysozyme (HEL₁₁₆₋₁₂₉, accession # LZCH) monoclonalantibodies.

Antigen processing of bacterial antigens is complex and cell typedependent. Host immunity to bacteria requires both CD8 and CD4responses. In general, CD8 and CD4 represent separate arms of the immuneresponse. CD8 cells represent the cellular immune response and CD4 cellsrepresent the humoral (antibody) immune response. Antigens thatstimulate these responses are processed differently by the host cell.Since there is more than one pathway for bacterial antigens to beprocessed, it makes sense that a better understanding of host immunitycould be acquired by determining the accessibility of bacterial antigenswithin each pathway. As such, tools can be designed for use in methodsof studying antigen processing within the class-II MHC pathway. Suchmethods allow the construction of more effective vaccines by allowingthe recruitment of carrier proteins to deliver antigens to the class-IIMHC pathway. The methods are performed similarly to the experimentsdetailed above for the class-I MHC pathway, except that MHC II nucleicacid sequence is included in the transposable element, and a MHC IIspecific binding agent is used in the assay.

EXAMPLE 11 Construction of Other Heterologous Antigen Vaccines

By disseminating epitopes throughout the genome of Salmonella, potentvaccines can be constructed by identification and use of carrierproteins that elicit protective immune responses. Salmonella causes adisseminated infection in several different tissues. The transposableelements of the present invention can be used to identify genesexpressed in different tissues, and vaccines can be constructed whichtailor the immune response by using tissue-specific carrier proteins ascarriers.

EXAMPLE 12 Alternate Transposable Elements

Fluorescent Protein Insertions.

Variants of the Tn5-DICE transposon and the DICE-I and DICE-IItransposomes can be constructed to carry one or more genes that encodefluorescent proteins. In -frame insertions of this transposon into agene will generate a fusion protein that carries an enhanced fluorescentprotein, for example GFP (accession U55761), and red fluorescent protein(accession U70496). As used herein, GFP refers to both the wild-typeprotein, and spectrally shifted mutants thereof, for example asdescribed in Tsien, 1998, Ann. Rev. Biochem. 67:509 and in U.S. Pat.Nos. 5,777,079 and 5,625,048 to Tsien and Heim, herein incorporated byreference. Asparyginyl endopeptidase cleavage sites enable thefluorescent protein to be cleaved from the fusion product eliminatingconformational distortions and allow the protein to fluoresce. The GFPgene would be placed within the same location as the I-A^(b) restrictedT-cell epitope, ASFEAQGALANIAVDKA contained in DICE-II. The GFP or RFPgenes would be modified to remove termination signals to allowtranscriptional and translational readthrough after insertion andresolution.

Addition of one or more fluorophores may allow the host bacterial rangeof the system to be greatly expanded, because it would enable theidentification in vivo of expressed genes by FACS analysis of tissuehomogenates as described in EXAMPLE 2. Protein products identified bythis transposon/transposome variant can be use to identify efficaciousbacterial vaccine antigens in previously genetically intractablemicroorganisms that are pathogenic to humans and animals. Thesetransposon/transposome variants can be used to identify secretedbacterial antigens by direct sorting of infected fluorescent host cells.

Customized Effector Proteins.

Variants of the Tn5-transposon and the DICE-I and DICE-II transposomescan be generated to engineer bacterial carrier vaccines to delivercustomized host effector molecules. For instance, by delivering afragment or an entire host signaling factor into the host cell afteruptake of the vaccine, the immune response could thus be skewed to amore efficacious response. Candidate signaling molecules include, butare not limited those in the Jak/Stat pathway.

Vaccines having the ability to appropriately bias the immune responseavoid many of the deleterious side effects associated with traditionalvaccines. In addition, vaccines can be constructed to enable thetreatment of acute pathogenic infections. The response to these types ofvaccines would be quick, strong, specific, and transient These types ofvaccines are desired by the armed forces as a means of dealing withbio-warfare exposure.

Multivalent Vaccines.

A variant of the vaccines described in the above examples that deliversepitopes from more than one organism can be generated. Salmonella can beused to construct multivalent vaccines since it is capable of carryinglarge amounts of accessory DNA encoding vaccine antigens. The strengthof the DICE system lies in its ability to identify appropriate carrierproteins for combinations of epitopes.

Many pathogenic infections potentiate the growth of additionalmicroorganisms that are different from the primary infection. Suchvaccines can be used as a “one shot” method of protection.

Host Receptor Delivey.

Variants of the transposon/transposases that deliver a molecule thatwill localize to the surface of the host cell can be generated. Suchconstructs have at least two potential uses. First, they would allowsecreted bacterial proteins to be identified after infection by lookingfor the presence of the secreted protein on the surface of the hostcell. Second, these variants could deliver chimeric signaling molecules(molecules which associate to the cell surface and initiate internalsignaling in response to an external signal). For example, deliveringthe vaccine then subsequently activating the response after treatmentwith a drug. This would allow antigen to be loaded into an APC and thusaugment the immune response.

Alpha-Omega Complementation.

Variants of the transposon/transposome that encode the α-fragment fromβ-galactosidase can be generated. Many bacteria are not amenable to theanalysis of secreted proteins because tools are not available that allowthe identification of secreted genes by their MHC-restriction. Thistransposon/transposome variant will enable the bacteria to secretefusion proteins that contain the α-fragment from β-galactosidase.Secreted proteins can be detected because the host cell (or a transgenichost animal) expresses the omega fragment from β-galactosidase. When thesecreted α-fragment and the host omega fragment come into contact toform a functional β-galactosidase complex, various enzyme substrates canbe used to visualize the interaction. For instance, the substrate C₁₂FDG(Molecular Probes, #1-2904) becomes fluorescent when cleaved byfunctional β-galactosidase. Alternatively, the commonly used substrateX-gal could be used to visualize active β-galactosidase within a cell.With system, pathogenesis can be studied in whole animals by looking forthe presence of fluorescent bacteria in different host tissues. Inaddition, tissue-specific secretion of bacterial proteins could bedetermined and thus enable optimized carrier vaccines that secreteantigen in appropriate host compartments.

EXAMPLE 13 Other Uses of Transposable Elements

The transposable elements of the present invention can also be used tomodify vaccine carrier strains of Salmonella to augment or skew theimmune response to the carried antigen by delivering eukaryotic effectorproteins such as Jak2 or Tyk2 as CAP fusions. Mutants generated by thetransposable elements can be used to identify tissue-specific SalmonellaCAPs, potentially useful proteins for regulating the timing of theimmune response to carried antigens and thus generate immune responsesmore amenable to the lifecycle of different pathogens. For instance,JAK2 (a host kinase) initiates a

signaling cascade that ultimately results in the upregulation ofcytokines that enhance the cell-mediated immune response. In principle,the transposon could be engineered to deliver JAK2 (or a portion ofJAK2) and bias the immune response to one that is predominately cellmediated.

EXAMPLE 14 Functional Genomics

Genomic sequencing of pathogens provides valuable insights into thelifestyle of a variety of different organisms. Data from these projectshowever reveal that as much as 40% of genes have no known function.Therefore, methods are needed to rapidly assign function to genesidentified by genomic projects. Since the transposable elements of thepresent invention can be constructed to carry an affinity tag such as a6× histidine site, immunolocalization studies can provide valuableinsight into the function of genes identified by genomic sequencingprojects.

EXAMPLE 15 Construction or Customized Effector Molecules

Specific immune responses are generated as a consequence of a cascade ofsignal transduction events. DICE identifies proteins that have access tothe cytoplasm of the host cell. DICE technology can be used to constructcustomized effector molecules whose function would be to skew the immuneresponse and generate a bacterial carrier vaccine appropriate toclearance of the pathogen.

EXAMPLE 16 Identification of Diagnostic Proteins

The emergence of new, more virulent bacterial strains, coupled with thethreat of biological terrorism, emphasizes the need for targets thatwill allow the rapid and precise identification of different pathogens.DICE enable the identification of species-specific genes utilized by thepathogen during the course of infection.

EXAMPLE 17 Transfer of DNA into Cells

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. The vectors areintroduced into the recipient cells as pure DNA (transfection) by, forexample, precipitation with calcium phosphate (Graham and vander Eb,1973, Virology 52:466) or strontium phosphate (Brash et al., 1987, Mol.Cell Biol. 7;2013), electroporation (Neumann et al., 1982, EMBO J.1:841), lipofection (Felgner et al., 1987, Proc. Nail. Acad Sci USA84:7413), DEAE dextran (McCuthan et al., 1968, J. Natl. Cancer Inst.41:351), microinjection (Mueller et al., 1978, Cell 15:579), protoplastfusion (Schafner, 1980, Proc. Nail. Acad. Sci. USA 77:2163-7), or pelletguns (Klein et al., 1987, Nature 327:70). Alternatively, the cDNA can beintroduced by infection with virus vectors. Systems are developed thatuse, for example, retroviruses (Bernstein et al., 1985, Gen. Engrg.7:235), adenoviruses (Ahmad et al., 1986, J. Virol. 57:267), or Herpesvirus (Spaete et al., 1982, Cell 30:295).

EXAMPLE 18 Sequence Variants of Transposable Elements

Having presented a format of the transposable elements of the presentinvention, and the sequence of DICE-I and DICE-II, this invention nowalso facilitates the creation of DNA molecules, and thereby proteins,which are derived from those disclosed but which vary in their precisenucleotide or amino acid sequence from those disclosed. Such variantsmay be obtained through a combination of standard molecular biologylaboratory techniques and the nucleotide sequence information disclosedby this invention.

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR.

Variant DNA molecules include those created by standard DNA mutagenesistechniques, for example, M13 primer mutagenesis. Details of thesetechniques are provided in Sambrook et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 15, hereinincorporated by reference). By the use of such techniques, variants maybe created which differ in minor ways from those disclosed. DNAmolecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and which differ from those disclosed bythe deletion, addition or substitution of nucleotides while stillencoding a protein which possesses the functional characteristics of theproteins which are comprehended by this invention.

Also within the scope of this invention are small DNA molecules whichare derived from the disclosed DNA molecules. Such small DNA moleculesinclude oligonucleotides suitable for use as hybridization probes or PCRprimers. As such, these small DNA molecules will include at least asegment of the transposable element DNA molecules and, for the purposesof PCR, will include at least 20-50 consecutive nucleotides of thetransposable element nucleic acid sequences. DNA molecules andnucleotide sequences which are derived from the disclosed DNA moleculesas described above may also be defined as DNA sequences which hybridizeunder stringent conditions to the DNA sequences disclosed, or fragmentsthereof.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989 ch. 9 and 11), hereinincorporated by reference. By way of illustration only, a hybridizationexperiment may be performed by hybridization of a DNA molecule (forexample, a deviation of the transposable element) to a target DNAmolecule (for example, a transposable element DNA) which has beenelectrophoresed in an agarose gel and transferred to a nitrocellulosemembrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), atechnique well known in the art and described in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989).

Hybridization with a target probe labeled with [³²P]-dCTP is generallycarried out in a solution of high ionic strength such as 6×SSC at atemperature that is 20-25° C. below the melting temperature, T_(m),described below. For such Southern hybridization experiments where thetarget DNA molecule on the Southern blot contains 10 ng of DNA or more,hybridization is typically carried out for 6-8 hours using 1-2 ng/mlradiolabeled probe (of specific activity equal to 10⁹ CPM/μg orgreater). Following hybridization, the nitrocellulose filter is washedto remove background hybridization. The washing conditions should be asstringent as possible to remove background hybridization but to retain aspecific hybridization signal. The term T_(m) represents the temperatureabove which, under the prevailing ionic conditions, the radiolabeledprobe molecule will not hybridize to its target DNA molecule. The T_(m)of such a hybrid molecule may be estimated from the following equation(Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390, 1962):T_(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)*(600/1);where I=the length of the hybrid in base pairs.

This equation is valid for concentrations of Na⁺ in the range of 0.01 Mto 0.4 M, and it is less accurate for calculations of T_(m) in solutionsof higher [Na⁺]. The equation is also primarily valid for DNAs whose G+Ccontent is in the range of 30% to 75%, and it applies to hybrids greaterthan 100 nucleotides in length (the behavior of oligonucleotide probesis described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

Thus, by way of example, for a 150 base pair DNA probe derived from atransposable element nucleic acid sequence (with a hypothetical %GC=45%), a calculation of hybridization conditions required to giveparticular stringencies may be made as follows: For this example, it isassumed that the filter will be washed in 0.3×SSC solution followinghybridization, thereby: [Na⁺]=0.045 M; % GC=45%; Formamideconcentration=0; I=150 base pairs;T_(m)=81.5−16.6(log₁₀[Na⁺])+(0.41×45)−(600/150); and so T_(m)=74.4° C.

The T_(m) of double-stranded DNA decreases by 1-1.5° C. with every 1%decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973).Therefore, for this given example, washing the filter in 0.3×SSC at59.464.4° C. will produce a stringency of hybridization equivalent to90%; that is, DNA molecules with more than 10% sequence variationrelative to the target transposable element DNA will not hybridize.Alternatively, washing the hybridized filter in 0.3×SSC at a temperatureof 65.4-68.4° C. will yield a hybridization stringency of 94%; that is,DNA molecules with more than 6% sequence variation relative to thetarget transposable element DNA molecule will not hybridize. The aboveexample is given entirely by way of theoretical illustration. Oneskilled in the art will appreciate that other hybridization techniquesmay be utilized and that variations in experimental conditions willnecessitate alternative calculations for stringency.

In particular embodiments of the present invention, stringent conditionsmay be defined as those under which DNA molecules with more than 25%,15%, 10%, 6% or 2% sequence variation (also termed “mismatch”) will nothybridize.

The degeneracy of the genetic code further widens the scope of thepresent invention as it enables major variations in the nucleotidesequence of a DNA molecule while maintaining the amino acid sequence ofthe encoded protein. For example, the C-terminal amino acid residue ofthe transposable element Tn5-DICE is alanine. This is encoded in theTn5-DICE DNA by the nucleotide codon triplet GCG. Because of thedegeneracy of the genetic code, other nucleotide codon triplets, couldencode the C-terminal amino acid residue (e.g. GCT and GCC), as theyalso code for alanine. Thus, the nucleotide sequence of the Tn5-DICEcDNA could be changed at this position to any of these three codonswithout affecting the amino acid composition of the encoded protein orthe characteristics of the protein. Based upon the degeneracy of thegenetic code, variant DNA molecules may be derived from the cDNAmolecules disclosed herein using standard DNA mutagenesis techniques asdescribed above, or by synthesis of DNA sequences. DNA sequences whichdo not hybridize under stringent conditions to the cDNA sequencesdisclosed by virtue of sequence variation based on the degeneracy of thegenetic code are herein also comprehended by this invention.

The invention also includes DNA sequences that are substantiallyidentical to any of the DNA sequences disclosed herein, wheresubstantially identical means a sequence that has identical nucleotidesin at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the alignedsequences.

One skilled in the art will recognize that the DNA mutagenesistechniques described above may be used not only to produce variant DNAmolecules, but will also facilitate the production of proteins whichdiffer in certain structural aspects from the transposable elements, yetwhich proteins are clearly derivative of this protein and which maintainthe essential characteristics of the proteins of the transposableelements. Newly derived proteins may also be selected in order to obtainvariations on the characteristic of the transposable element protein, aswill be more fully described below. Such derivatives include those withvariations in amino acid sequence including minor deletions, additionsand substitutions.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed protein variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence as described aboveare well known.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of about from 1 to 10 amino acid residues;and deletions will range about from 1 to 30 residues. Deletions orinsertions may be made in adjacent pairs, i.e., a deletion of 2 residuesor insertion of 2 residues. Substitutions, deletions, insertions or anycombination thereof may be combined to arrive at a final construct.Obviously, the mutations that are made in the DNA encoding the proteinmust not place the sequence out of reading frame and ideally will notcreate complementary regions that could produce secondary mRNAstructure.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions generally are made conservatively, asdefined above.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those definedabove, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in proteinproperties will be those in which (a) a hydrophilic residue, e.g., serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

The effects of these amino acid substitutions or deletions or additionsmay be assessed for derivatives of the transposable elements by assaysin which the ability of the elements to transpose are assessed.

EXAMPLE 19 Pharmaceutical Compositions and Modes of Administration

Various delivery systems for administering the transposable elements ofthe present invention are known, and include e.g., encapsulation inliposomes, microparticles, microcapsules, expression by recombinantcells, receptor-mediated endocytosis (see Wu and Wu, J. Biol. Chem.1987, 262:4429-32), and construction of a therapeutic nucleic acid aspart of a retroviral or other vector. Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, and oral routes. The compoundsmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, the pharmaceuticalcompositions may be introduced into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

In one embodiment, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment,for example, by local infusion during surgery, topical application,e.g., in conjunction with a wound dressing after surgery, by injection,through a catheter, by a suppository ran implant, such as a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers. In one embodiment, administration can beby direct injection at the site (or former site) of a malignant tumor orneoplastic or pre-neoplastic tissue.

The use of liposomes as a delivery vehicle is one delivery method ofinterest. The liposomes fuse with the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the target cells for a sufficient time for fusion to occur,using various means to maintain contact, such as isolation and bindingagents. Liposomes may be prepared with purified proteins or peptidesthat mediate fusion of membranes, such as Sendai virus or influenzavirus. The lipids may be any useful combination of known liposomeforming lipids, including cationic lipids, such as phosphatidylcholine.Other potential lipids include neutral lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like. For preparingthe liposomes, the procedure described by Kato et al. (J Biol. Chem.1991, 266:3361) may be used.

The present invention also provides pharmaceutical compositions whichinclude a therapeutically effective amount of the transposable element,alone or with a pharmaceutically acceptable carrier. In one example,homogeneous compositions of transposable element therapeutic moleculesincludes compositions that are comprised of at least 90°% of thepeptide, variant, analog, derivative or mimetic in the composition.

Delivery System

Such carriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile, and the formulation suits themode of administration. The composition can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, and magnesiumcarbonate.

The amount of the inducing agent and disrupting agent that will beeffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach subject's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more often ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture , use or sale for human administration. Instructions for useof the composition can also be included.

The pharmaceutical compositions or methods of treatment may beadministered in combination with other therapeutic treatments, such asother antineoplastic or antitumorigenic therapies.

Administration of Nucleic Acid Molecules

In an embodiment in which a transposable element nucleic acid isemployed for gene delivery or therapy, the analog is deliveredintracellularly (e.g., by expression from a nucleic acid vector or byreceptor-mediated mechanisms). In a specific embodiment where thetherapeutic molecule is a nucleic acid or antisense molecule,administration may be achieved by an appropriate nucleic acid expressionvector which is administered so that it becomes intracellular, e.g., byuse of a retroviral vector (see U.S. Pat. No. 4,980,286), or by directinjection, or by use of microparticle bombardment (e.g., a g ene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., Proc. Natl. Acad Sci. USA 1991, 88:1864.8).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

The vector pcDNA, is an example of a method of introducing the foreigncDNA into a cell under the control of a strong viral promoter (CMV) todrive the expression. However, other vectors can be used. Otherretroviral vectors (such as pRETRO-ON, Clontech), also use this promoterbut have the advantages of entering cells without any transfection aid,integrating into the genome of target cells only when the target cell isdividing (as cancer cells do, especially during first remissions afterchemotherapy) and they are regulated. It is also possible to turn on theexpression of the transposable element nucleic acid by administeringtetracycline when these plasmids are used. Hence these plasmids can beallowed to transfect the cells, then administer a course of tetracyclinewith a course of chemotherapy to achieve better cytotoxicity.

Other plasmid vectors, such as pMAM-neo (Clontech) or pMSG (AmershamPharmacia Biotech, Piscataway, N.J.) use the MMTV-LTR promoter (whichcan be regulated with steroids) or the SV10 late promoter (pSVL,Amersham Pharmacia Biotech, Piscataway, N.J.) ormetallothionein-responsive promoter (pBPV, Amersham Pharmacia Biotech)and other viral vectors, including retroviruses. Examples of other viralvectors include adenovirus, AAV (adeno-associated virus), recombinantHSV, poxviruses (vaccinia) and recombinant lentivirus (such as HIV). Allthese vectors achieve the basic goal of delivering into the target cellthe cDNA sequence and control elements needed for transcription. Thepresent invention includes all forms of nucleic acid delivery, includingsynthetic oligos, naked DNA, plasmid and viral, integrated into thegenome or not.

Having illustrated and described the principles of constructing andusing transposable elements, it should be apparent to one skilled in theart that the invention can be modified in arrangement and detail withoutdeparting from such principles. In view of the many possible embodimentsto which the principles of our invention may be applied, it should berecognized that the illustrated embodiments are only examples of theinvention and should not be taken as a limitation on the scope of theinvention. Rather, the scope of the invention is in accord with thefollowing claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A method for identifying a protein secreted by an intracellular pathogen and having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen, comprising: (i) transfecting intracellular pathogen with a transposable element, wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class I epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen; (ii) transforming the intracellular pathogen with a vector comprising a transposase; (iii) contacting a eukaryotic cell that can internalize the intracellular pathogen with the pathogen transfected with the transposable element, wherein an MHC class I haplotype of the eukaryotic cell is matched to the MHC I epitope; (iv) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class I epitope, thereby generating a labeled eukaryotic cell; (v) identifying the labeled eukaryotic cell; (vi) lysing the labeled eukaryotic cell to externalize the intracellular pathogen; (vii) growing the externalized intracellular pathogen to produce a population of intracellular pathogen; and (viii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class I pathway of eukaryotic cell infected with the intracellular pathogen.
 2. The method of claim 1, wherein the eukaryotic cell is a cell of the immune system.
 3. The method of claim 2, wherein the cell of the immune system is a macrophage.
 4. The method of claim 1, wherein the identification of the labeled eukaryotic cell is by fluorescence activated cell sorting.
 5. The method of claim 1, wherein the intracellular pathogen is a bacterial cell.
 6. The method of claim 1, wherein the pathogen is Salmonella, Mycobacterium tuberculosis, Plasmodium, or Listeria monocytogenes.
 7. The method of claim 1, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a fit recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.
 8. The method of claim 1, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.
 9. The method of claim 8, wherein the loxP sequence comprises the sequence shown in SEQ ID NO:
 11. 10. The method of claim 1, wherein the MHC class I epitope is SIINFEKL (SEQ ID NO: 6) and the MHC class I haplotype of the eukaryotic cell is H-2 Kb.
 11. The method of claim 1, wherein the selectable marker is a nucleic acid encoding antibiotic resistance.
 12. The method of claim 11, wherein the antibiotic resistance is ampicillin, kanamycin, zeomycin, hygromycin, tetracycline, puromycin or bleomycin resistance.
 13. The method of claim 1, wherein the selectable marker is detected by spectrophotometric properties.
 14. The method of claim 1, wherein the selectable marker is beta-galactosidase or green fluorescent protein.
 15. The method of claim 1, wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO:
 5. 16. The method of claim 15, wherein the insertion end at the 5′ end of the transposable element comprises the sequence shown in SEQ ID NO:
 5. 17. The method of claim 1, wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO: 3 or SEQ ID NO:
 4. 18. The method of claim 17, wherein the insertion end at the 3′ end of the transposable element comprises the sequence shown in SEQ ID NO:
 3. 19. The method of claim 1, wherein the transposable element further comprises a nucleic acid sequence encoding a transposase.
 20. The method of claim 19, wherein the transposase is a Cre transposase.
 21. The method of claim 1, wherein the transposable element further comprises an affinity tag.
 22. The method of claim 21, wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.
 23. The method of claim 22, wherein the affinity tag is 6× histidine.
 24. The method of claim 21, wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.
 25. The method of claim 21, wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.
 26. The method of claim 1, wherein the MHC class I epitope is LLFGYPVYV (SEQ ID NO: 7) and the MHC class I haplotype of the eukaryotic cell is HLA-A2.
 27. A method for identifying a protein secreted by an intracellular pathogen and having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen, comprising: (i) transfecting an intracellular pathogen expressing a tranposase with a transposable element wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class I epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen; (ii) contacting a eukaryotic cell that can internalize the intracellular pathogen with the pathogen transfected with the transposable element, wherein an MHC class I haplotype of the eukaryotic cell is matched to the MHC I epitope; (iiii) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class I epitope, thereby generating a labeled eukaryotic cell; (iv) identifying the labeled eukaryotic cell; (v) lysing the labeled eukaryotic cell to externalize the intracellular pathogen; (vi) growing the externalized pathogen to produce a population of intracellular pathogen; and (vii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen.
 28. The method of claim 27, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a fit recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.
 29. The method of claim 28, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.
 30. The method of claim 29, wherein the loxP sequence comprises the sequence shown in SEQ ID NO:
 11. 31. The method of claim 27, wherein the MHC class I epitope is SIINFEKL (SEQ ID NO: 6) and the MHC class I haplotype of the eukaryotic cell is H-2 Kb.
 32. The method of claim 27, wherein the selectable marker is a nucleic acid encoding antibiotic resistance.
 33. The method of claim 27, wherein the selectable marker is detected by spectrophotometric properties.
 34. The method of claim 27, wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO:
 5. 35. The method of claim 27, wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO; 3 or SEQ ID NO:
 4. 36. The method of claim 27, wherein the transposable element further comprises an affinity tag.
 37. The method of claim 36, wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.
 38. The method of claim 36, wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.
 39. The method of claim 36, wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.
 40. The method of claim 27, wherein the MHC class I epitope is LLFGYPVYV (SEQ ID NO: 7) and the MHC class I haplotype of the eukaryotic cell is HLA-A2.
 41. A method for identifying a protein secreted by an intracellular pathogen and having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen, wherein the intracellular pathogen is a bacterial cell, comprising: (i) transfecting an intracellular pathogen with a transposable element, wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class I epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and a transposase, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen; (ii) contacting a eukaryotic cell that can internalize the intracellular pathogen with the pathogen transfected with the transposable element, wherein an MHC class I haplotype of the eukaryotic cell is matched to the MHC I epitope; (iii) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class I epitope, thereby generating a labeled eukaryotic cell; (iv) identifying the labeled eukaryotic cell; (v) lysing the labeled eukaryotic cell to externalize the intracellular pathogen; (vi) growing the externalized bacterial cell to produce a population of intracellular pathogen; and (vii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen.
 42. The method of claim 41, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a flt recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.
 43. The method of claim 42, wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.
 44. The method of claim 43, wherein the loxP sequence comprises the sequence shown in SEQ ID NO:
 11. 45. The method of claim 41, wherein the MHC I epitope is SIINFEKL (SEQ ID NO: 6) and the MHC class I haplotype of the eukaryotic cell is H-2 Kb.
 46. The method of claim 41, wherein the selectable marker is a nucleic acid encoding antibiotic resistance.
 47. The method of claim 41, wherein the selectable marker is detected by spectrophotometric properties.
 48. The method of claim 41, wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO:
 5. 49. The method of claim 41, wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO: 3 or SEQ ID NO:
 4. 50. The method of claim 46, wherein the transposable element further comprises an affinity tag.
 51. The method of claim 50, wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.
 52. The method of claim 50, wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.
 53. The method of claim 50, wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.
 54. The method of claim 41, wherein the MHC class I epitope is LLFGYPVYV (SEQ ID NO: 7) and the MHC class I haplotype of the eukaryotic cell is HLA-A2. 